The specific aims of this application are: 1) To evaluate the role of a high-affinity calcium-activated ATPase in the renal tubular transport of calcium, 2) To identify the nephron segments which contain the specific binding sites for glucocorticoids and insulin and to study therein the effect of these hormones on membrane Na-K-ATPase activity and kinetic properties. In the proposed research we will take advantage of micromethods developed by us which permit localization of physiologic and biochemical events in discrete segments of the nephron. In the first part of the study we will examine the proposition that a membrane bound ATPase activated by free Ca++ in micromolar concentrations (such as are present in the cytosol) participates in the tubular reabsorption of this cation. We will measure the activity of Ca-Mg-ATPase in purified preparations of luminal and basolateral tubular cell membranes, and will evaluate in isolated tubules the effects upon this enzyme of various hormones known to influence the tubular reabsorption of calcium; in addition, we will study calmodulin, an activator of the plasma membrane calcium-ATPase in other tissues, but whose function in the kidney is at present unknown. These studies will be performed both in vitro, by pretreating membranes or isolated tubules with the effector, and in vivo, by measuring the activity and properties of Ca-Mg-ATPase in animals treated with the various hormones. The studies dealing with the interactions of glucocorticoids and insulin with the kidney have a dual long term objective: a) To map the distribution along the nephron of binding sites for hormones which do not have cyclic AMP as their second messenger, since identification of tubular target sites has been possible to date only for hormones that stimulate adenylate cyclase and b) To evaluate the mechanisms that mediate the effects of glucocorticoids and insulin on sodium and potassium transport by the renal tubule. Information on the latter two topics is at present fragmentary and inconclusive, mainly because of the difficulties involved in the identification of the nephron segments that represent target sites of these hormones. This limitation can be circumvented by the use of the microtechniques proposed in this application.